The present invention relates to methods and compositions for immunoassays, and to kits for carrying out such methods and containing such compositions. The invention more specifically relates to immunoassays in which at least three receptors are employed to capture and insolubilize a sample analyte.
Receptors are generally proteins, although other substances having reversible specific binding affinity for a ligand or for a labeled analogue thereof are equally useful. The most commonly used receptors are antibodies because they can be raised to bind any desired ligand. However, other binding proteins are included within the meaning of receptor, for example, avidin, streptavidin, lectins, enzymes, apoenzymes, hydrophobic protein binding sites, intrinsic factor, thyroxine binding globulin, cortisol binding protein, folate binding protein, and membrane-associated specific receptor proteins. Receptors may also include polynucleic acid sequences and low molecular weight substances, such as, dyes or biotin. As a general rule, receptors are the larger of the two components of a binding pair. However, for the purposes of the present invention, a receptor is to be construed as simply one component of a binding pair.
Ligands are the converse of receptors. Whereas ligands are ordinarily low molecular weight organic compounds, they are defined for purposes of this invention, as one component of a binding pair, a receptor being the other component, wherein the ligand may be larger or smaller than the receptor.
Labeled ligand analogues are derivatives of ligands which carry a detectable substituent, such as, an enzyme, radioisotope, stable free radical or other known label, but which are recognizable by and bind to a receptor for the parent ligand (sample analyte) with approximately the same affinity as exhibited by the parent ligand.
A ligand-receptor pair is by definition reversibly bindable, that is, its behavior is governed by the law of mass action. On a practical level, "reversibly bindable" indicates that either component of the ligand-receptor pair is displaceable by structurally related substances under the conditions normally encountered in immunoassays, that is, mild pH and temperature, and moderate ionic strength.
Immunoassays employing double receptors are known in the art. Such double receptor immunoassays have as their salient feature a first receptor specific for a second receptor wherein the second receptor ordinarily binds to the ligand (analyte) which is the substance which is to be determined in the assay. Exemplary of such a double receptor immunoassay is that disclosed in U.S. Pat. No. 4,343,896 (issued Aug. 10, 1982 to Wolters and Kuypers) wherein a sandwich immunocomplex is noncovalently bonded to a solid phase through immobilized polyclonal antibodies raised against one of the antibodies of the sandwich.
The double receptor methods (without the use of binding ligands) have a number of disadvantages, principally because the affinity of such receptors for one another is not sufficiently high. Such limitations are manifested in requirements for relatively large plastic or solid surfaces for adsorption of receptor, making it difficult to use such a format in a coated tube, and for prolonged incubations. The low affinities are critical when the receptor or the antigen used to raise the receptor for the sample ligand is difficult to obtain.
U.S. Pat. No. 4,271,140 (issued Jun. 2, 1981 to Bunting) provides one method of overcoming such nonspecificity of one receptor for another in a double receptor immunoassay context. That patent is based on the fact that the affinity of antibodies for binding ligands is high. (Wilchek et al., FEBS Letters., 31(1):149-152 (April, 1973); Abbas et al., J. Immunol, 11 4(4):1197-1204 (April 1975); Lamm et al., PNAS (USA), 69(12):3732-3736 (December, 1972); and Wofsy et al., J, Exp. Med., 140:523-537 (1974) ) The Bunting patent claims an improvement to double receptor specific binding assays wherein instead of a double receptor, the assay comprises a receptor complex having a binding ligand (BL), a labeled or unlabeled receptor specific for the binding ligand (A.sub.BL) and a second receptor (A.sub.1). The configuration of the complex is A.sub.BL (BL)n A.sub.1 (wherein n is at least one), and BL is reversibly bound to A.sub.BL and covalently bound to A.sub.1. BL can be fluorescein isothiocyanate (FITC).
Another patent employing the concept of using a binding ligand, termed therein a hapten, to increase the specificity of one receptor for another in double receptor immunoassays is U.S. Pat. No. 4,659,678 (issued to Forrest et al. on Apr. 21, 1987). That patent claims an immunoassay method for an antigen comprising incubating a mixture of: (a) a liquid sample; (b) labeled monoclonal antibodies to the antigen; (c) monoclonal antibodies to the antigen which are conjugated with a hapten; and (d) an antibody raised to the hapten which non-covalently bonds to the hapten and which is bound to a solid phase support. The hapten can be fluorescein isothiocyanate. That patent further discloses at column 5, lines 14-18, an immunoassay method wherein a specific binding protein and its corresponding ligand, for example, avidin and biotin, are respectively bound to a solid phase and conjugated to an antibody to the antigen under assay and by the noncovalent bonding thereof effect the insolubilization of an immunocomplex of the antigen, a labeled antibody thereto, and the unlabeled antibody conjugated to the ligand of the specific binding pair.
Other exemplary variations of the double receptor immunoassay include the following three references.
European Patent Application Publication No. 337,082 (assigned to BioRad Lab. Inc.) describes an immunoassay wherein an anti-biotin antibody is immobilized on a solid support and incubated with a biotinylated binding species, which is able to bind a sample ligand, so as to produce a ligand-specific binding site on the support.
European Patent Application Publication No. 344,578 (assigned to Boehringer Mannheim GMBH) describes a heterogeneous immunoassay using a solid phase to which an immunological reactant is fixed wherein the new feature is that the solid phase used is a reaction vessel having fixed to its internal surfaces enough streptavidin or avidin to provide 0.1 to 2.5 micrograms/milliliter (.mu.tg/ml) of reaction volume. The reaction vessels are described as being useful for any assay in which the antibody or antigen which is to be immobilized can be conjugated with biotin.
U.S. Pat. No. 4,778,751 (issued to El Shami et al. on Oct. 18, 1988) claims a method for measuring the level of an analyte (an antigen, antibody or hapten) in a liquid sample which sequentially comprises: (a) forming a soluble complex wherein the analyte is linked through a specific antibody, antigen or anti-hapten depending on the analyte, to a soluble matrix which carries a ligand (X) and is capable of being chemically attached to more than one specific antibody, antigen or antihapten; (b) forming an insolubilized complex comprising a solid support linked to the ligand (X) of the matrix through an anti-ligand (Y) wherein the insolubilized complex carries a label (Z) linked to the analyte through an anti-antigen, anti-antibody or anti-hapten; (c) washing the insolubilized complex; and (d) observing the presence of label. The ligand (X) can be biotin; and the anti-ligand (Y) can be avidin. The soluble matrix can be a soluble carbohydrate, dextran or a soluble polymer.
The El Shami et al. patent further discloses at column 21, lines 34-68 and column 22, lines 1-5, variations of said method wherein it is desired to capture the analyte in a liquid phase and then immobilize the soluble matrix in the same reaction. Three possible configurations are disclosed as follows: (1) X and Y are both avidin; and biotin is added after the initial liquid phase reaction has been completed; (2) X is biotin, Y is antiavidin and avidin is added after the liquid phase reaction; and (3) X is biotin, Y is a biotinylated protein, and avidin is added after the liquid phase reaction.
The novel immunoassay system of the present invention provides a capture system comprising at least three receptors. The first two receptors constitute a generic capture system applicable to a variety of immunoassay formats for any analyte. The first receptor is bound to a solid phase and has as its ligand a second receptor or a ligand conjugated to said second receptor, wherein said second receptor is a bridge receptor, preferably an anti-hapten bridge receptor. The second receptor is termed a bridge receptor in that it provides a bridge between an immunocomplex formed freely in solution and the first receptor bound to the solid phase. The bridge receptor therefore separates the immunocomplex containing the analyte under assay from the liquid phase. The bridge receptor binds to a ligand conjugated to a sample analyte receptor, the third receptor. The third receptor has as its ligand either the analyte under assay or a further receptor which has as its ligand the analyte under assay.
It is an object of the present invention to provide a novel immunoassay methodology which not only overcomes the problem of nonspecificity of the noncovalent bonding between receptors of prior art double receptor immunoassays but also provides for the advantages of liquid phase kinetics and lack of steric hindrance in forming an immunocomplex.
It is further an object of the present invention to provide a universal solid phase and capture system which can be employed for competitive, sandwich, competitive/sandwich and related immunoassays for any analyte.
It is a further object of this invention to provide for a generic capture system, which is stable, has a long shelf life, and provides a solid phase component which has a first receptor uniformly bound to it. The generic capture system provides a solid phase coated with a first receptor, such as avidin or streptavidin, and a bridge receptor, such as a biotinylated anti-hapten antibody, which can be used irrespective of the analyte under assay.
It is a still further object of the invention to provide a solid phase uniformly coated with the first receptor that can be used with a variety of bridge receptors, for example, a variety of biotinylated antibodies raised against different haptens. Thus, if, for example, there is a concern that a particular hapten might represent a cross-reactivity problem when a particular analyte is to be assayed, a more preferred bridge receptor raised against a different hapten can be selected.
In preferred embodiments of this invention, options are provided wherein it is unnecessary to immobilize antibodies to the solid phase, for example, wherein avidin or more preferably streptavidin is the first receptor, and a very uniform, stable and consistent coating of the solid phase can be produced.
It is still further an object of the invention to provide for an immunocomplex capture step that can be driven to rapid completion by optimizing the amount of a generic bridge receptor that is added to the reaction mixture, preferably after the immunocomplex has been formed in solution.
Other objects of this invention will be apparent to those skilled in the art from a consideration of this specification and attached claims taken in their entirety.